Expression, purification, and characterization of iron-sulfur cluster assembly regulator IscR from Acidithiobacillus ferrooxidans.
IscR (iron-sulfur cluster regulator) has been reported to be a repressor of the iscRSUA operon, and in vitro transcription reactions have revealed that IscR has a repressive effect on the iscR promoter in the case of [Fe(2)S(2)] cluster loading. In the present study, the iscR gene from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, and then purified by one-step affinity chromatography to homogeneity. The molecular mass of the IscR was 18 kDa by SDS-PAGE. The optical and EPR spectra results for the recombinant IscR confirmed that an iron-sulfur cluster was correctly inserted into the active site of the protein. However, no [Fe(2)S(2)] cluster was assembled in apoIscR with ferrous iron and sulfide in vitro. Therefore, the [Fe(2)S(2)] cluster assembly in IscR in vivo would appear to require scaffold proteins and follow the Isc "AUS" pathway.
Zeng
Jia
J
Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha 410083, P R China.
Zhang
Ke
K
Liu
Jianshe
J
Qiu
Guanzhou
G
D042763:Acidithiobacillus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009876:Operon; D016415:Sequence Alignment
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins